Enhanced expression in a plant plastid

ABSTRACT

Novel compositions and methods useful for genetic engineering of plant cells to provide increased expression in the plastids of a plant or plant cell of a protein which produces a phenotype which is present when the plant or plant cell is grown in the absence of means for selecting transformed cells. Expression of the Bacillus thuringiensis bacterial protoxin in a plant chloroplast is exemplified.

INTRODUCTION

This application is a continuation of application PCT/US95/02901 filedMar. 10, 1995 and a continuation-in-part of U.S. application Ser. No.08/209,649 filed Mar. 11, 1994 issued as U.S. Pat. No. 5,545,817.

FIELD OF THE INVENTION

This invention relates to the application of genetic engineeringtechniques to plants. More specifically, the invention relates tocompositions and methods for enhancing expression of a peptide ofinterest in the plastid of a plant cell.

BACKGROUND

Plastids of higher plants, i.e. chloroplasts, amyloplasts andchromoplasts, have the same genetic content, and thus are believed to bederived from a common precursor, known as a proplastid. The plastidgenome is circular and varies in size among plant species from about 120to about 217 kilobase pairs (kb). The genome typically includes a largeinverted repeat, which can contain up to about 76 kilobase pairs, butwhich is more typically in the range of about 20 to about 30 kilobasepairs. The inverted repeat present in the plastid genome of variousorganisms has been described (Palmer, J. D. (1990) Trends Genet.6:115-120).

One advantage of plant plastid transformation over nucleartransformation is that the plastids of most plants are maternallyinherited, and consequently heterologous plastid genes are not pollendisseminated. This feature is particularly attractive for transgenicplants having altered agronomic traits, as introduced resistance ortolerance to natural or chemical conditions will not be transmitted towild-type relatives.

Plant plastids are also major biosynthetic centers. In addition tophotosynthesis in chloroplasts, plastids are responsible for productionof important compounds such as amino acids, complex carbohydrates, fattyacids, and pigments.

Plastids can also express two or more genes from a single plastidpromoter region. A DNA sequence expressed in a plastid may thus includea number of individual structural gene encoding regions under control ofone set of regulatory components. Thus, it is possible to introduce andexpress multiple genes in a plant cell, either from an engineeredsynthetic sequence or from a pre-existing prokaryotic gene cluster.

Such an expression method makes possible large scale and inexpensiveproduction of certain proteins and fine chemicals that are notpractically produced through standard nuclear transformation methods. Innuclear expression from introduced genes, each encoding sequence must beengineered under the control of a separate regulatory region, i.e., amonocistron. As a consequence, gene expression levels vary widely amongintroduced sequences, and generation of a number of transgenic plantlines is required, with crosses necessary, to introduce all of thecistrons into one plant and to get proper coordinated expression in thetarget biochemical pathway.

Plastids can be present in a plant cell at a very high copy number, withup to 50,000 copies per cell present for the chloroplast genome(Bendich, A. J. (1987) BioEssays 6:279-282). Thus, through plastidtransformation plant cells can be engineered to maintain an introducedgene of interest at a very high copy number.

For all of the above reasons, the plastids of higher plants present anattractive target for genetic engineering. Stable transformation ofplastids has been reported in the green algae Chlamydomonas (Boynton etal. (1988) Science 240:1534-1538) and more recently in higher plants(Svab et al. (1990) Proc. Natl. Acad. Sci. USA 87:8526-8530: Svab andMaliga (1993) Proc. Natl. Acad. Sci. USA 90:913-917); (Staub, J. M. andMaliga, P. (1993), EMBO J. 12:601-606). The method disclosed for plastidtransformation in higher plants relies on particle gun delivery of DNAcontaining a selectable marker and targeting of the DNA to the plastidgenome through homologous recombination.

Many examples exist where expression levels greater than what ispossible from nuclear expression would be desirable. One example can befound in those instances where it is desired to produce a novelsubstance in a mature plant for subsequent extraction and purification.Other examples of proteins which may need to be expressed at very highlevels are those producing resistance or tolerance phenotypes in theplant. One example of such a phenotype is a toxin active against plantpests.

In particular, there is a continuing need to introduce newly discoveredor alternative Bacillus thuringiensis genes into crop plants. Cryproteins (d-endotoxins) from Bacillus thuringiensis have potentinsecticidal activity against a number of Lepidopteran, Dipteran, andColeopteran insects. These proteins are classified CryI to CryV, basedon amino acid sequence homology and insecticidal activity. Most CryIproteins are synthesized as protoxins (ca. 130-140 kDa) then solubilizedand proteolytically processed into active toxin fragments (ca. 60-70kDa).

The poor expression of the protoxin genes from the nucleus of plants hasheretofore required the use of `truncated` versions of these genes. Thetruncated versions code only for the active toxin fragments. Otherattempts to increase the expression efficiency have includedresynthesizing the Bacillus thuringiensis toxin genes to utilize plantpreferred codons. Many problems can arise in such extensivereconstruction of these large cry genes (approximately 3.5 Kb), and theprocess is both laborious and expensive.

Problems can also arise as new insect pests become endemic, or asexisting populations develop resistance to a particular level or type ofBacillus thuringiensis toxin. Thus, there is a particular need forproducing higher and thereby more effective levels of the Bacillusthuringiensis toxin in plants, a need which will only increase withtime.

SUMMARY OF THE INVENTION

By this invention, plastid expression constructs are provided which areuseful for genetic engineering of plant cells and which provide forenhanced expression of a foreign peptide in plant cell plastids. Thetransformed plastid is preferably a metabolically active plastid, suchas the chloroplasts found in green plant tissues including leaves orcotyledons. The plastid is preferably one which is maintained at a highcopy number in the plant tissue of interest.

The plastid expression constructs for use in this invention generallyinclude a plastid promoter region and a DNA sequence of interest to beexpressed in transformed plastids. The DNA sequence of interest may be asingle encoding region, or may contain a number of consecutive encodingregions, to be expressed as an operon, for example where introduction ofa foreign biochemical pathway into plastids is desired.

In one embodiment, the DNA encoding sequence of the construct encodesthe same amino acid sequence as the native DNA sequence, while having acodon usage enriched for adenine and thymine content. As an example, anative DNA sequence may be resynthesized to include an adenine andthymine content preferred by the plant plastid. While the adenine andthymine percentage content of the nuclear genome varies from organism toorganism, in plants the codon utilization generally comprises moreguanine and cytosine pairings than adenine and thymine, thus the contentis considered enriched for guanine plus cytosine.

Plastid expression constructs of this invention may be linked to aconstruct having a DNA sequence encoding a selectable marker which canbe expressed in a plant plastid. Expression of the selectable markerallows the identification of plant cells comprising a plastid expressingthe marker.

In a preferred embodiment, transformation vectors for transfer of theconstruct into a plant cell include means for inserting the expressionand selection constructs into the plastid genome. This preferablycomprises regions of homology to the target plastid genome which flankthe constructs.

Also by this invention a method is provided whereby a plastid expressionconstruct is used to produce a peptide of interest in a plant cell. Thepeptide may be expressed in a plastid of the plant cell from the nativeDNA encoding sequence to the peptide. Alternatively, the DNA encodingsequence of the construct can be one enriched for adenine and thymine.

By this invention the insecticidal Bacillus thuringiensis toxin isproduced in plastids of a plant cell from the native DNA encodingsequence, with enhanced levels of expression of an insect resistantphenotype, as measured by insect feeding assays. The native Bacillusthuringiensis DNA encoding sequence may be the truncated versionspecific to the active fragment. This invention also provides theexpression of the Bacillus thuringiensis toxin from the non-truncatedsequence which encodes the protoxin.

Plant cells and plants produced by a method of the invention andcomprising a plastid expression construct are also considered in thisinvention.

DESCRIPTION OF THE FIGURES

FIG. 1 shows integration of cry genes from vectors pZS223 and pZS224into the wild-type plastid genome (Nt-ptDNA) to yield transplastomesNt-pZS223 ptDNA and Nt-pZS224 ptDNA, respectively.

DETAILED DESCRIPTION OF THE INVENTION

A plastid expression construct of this invention generally comprises apromoter functional in a plant plastid, a DNA sequence encoding apeptide of interest and a transcription termination region capable ofterminating transcription in a plant plastid. These elements areprovided as operably joined components in the 5' to 3' direction oftranscription.

Any DNA encoding sequence which is enriched for adenine plus thyminecontent, and which can be inserted into the plastid genome of a plantcell to provide enhanced expression of a peptide of interest from theDNA encoding sequence, can be utilized.

In developing the constructs the various fragments comprising theregulatory regions and open reading frame may be subjected to differentprocessing conditions, such as ligation, restriction enzyme digestion,PCR, in vitro mutagenesis, linkers and adapters addition, and the like.Thus, nucleotide transitions, transversions, insertions, deletions, orthe like, may be performed on the DNA which is employed in theregulatory regions or the DNA sequences of interest for expression inthe plastids. Methods for restriction digests, Klenow blunt endtreatments, ligations, and the like are well known to those in the artand are described, for example, by Maniatis et al. (in Molecularcloning: a laboratory manual (1982) Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y.).

During the preparation of the constructs, the various fragments of DNAwill often be cloned in an appropriate cloning vector, which allows foramplification of the DNA, modification of the DNA or manipulation of theDNA by joining or removing sequences, linkers, or the like. Preferably,the vectors will be capable of replication to at least a relatively highcopy number in E. coli. A number of vectors are readily available forcloning, including such vectors as pBR322, vectors of the pUC series,the M13 series vectors, and pBluescript vectors (Stratagene; La Jolla,Calif.).

In order to provide a means of selecting the desired plant cells,vectors for plastid transformation typically contain a construct whichprovides for expression of a selectable marker gene. Marker genes areplant-expressible DNA sequences which express a polypeptide whichresists a natural inhibition by, attenuates, or inactivates a selectivesubstance, i.e., antibiotic, herbicide etc..

Alternatively, a marker gene may provide some other visibly reactiveresponse, i.e., may cause a distinctive appearance or growth patternrelative to plants or plant cells not expressing the selectable markergene in the presence of some substance, either as applied directly tothe plant or plant cells or as present in the plant or plant cell growthmedia.

In either case, the plants or plant cells containing such selectablemarker genes will have a distinctive phenotype for purposes ofidentification, i.e., they will be distinguishable from non-transformedcells. The characteristic phenotype allows the identification of cells,cell groups, tissues, organs, plant parts or whole plants containing theconstruct.

Detection of the marker phenotype makes possible the selection of cellshaving a second gene to which the marker gene has been linked. Thissecond gene typically comprises a desirable phenotype which is notreadily identifiable in transformed cells, but which is present when theplant cell or derivative thereof is grown to maturity, even underconditions wherein the selectable marker phenotype itself is notapparent.

The use of such a marker for identification of plant cells containing aplastid construct has been described. Svab et al. (1993 supra). In theexamples provided below, a bacterial aada gene is expressed as themarker under the regulatory control of chloroplast 5' promoter and 3'transcription termination regions, specifically the tobacco 16S rRNApromoter rrn region and rps16 3' termination region. Numerous additionalpromoter regions may also be used to drive expression of the selectablemarker gene, including various plastid promoters and bacterial promoterswhich have been shown to function in plant plastids.

Expression of the aadA gene confers resistance to spectinomycin andstreptomycin, and thus allows for the identification of plant cellsexpressing this marker. The aadA gene product allows for continuedgrowth and greening of cells whose chloroplasts comprise the selectablemarker gene product. Cells which do not contain the selectable markergene product are bleached. Selection for the aada gene marker is thusbased on identification of plant cells which are not bleached by thepresence of streptomycin, or more preferably spectinomycin, in the plantgrowth medium.

A number of markers have been developed for use with plant cells, suchas resistance to chloramphenicol, the aminoglycoside G418, hygromycin,or the like. Other genes which encode a product involved in chloroplastmetabolism may also be used as selectable markers. For example, geneswhich provide resistance to plant herbicides such as glyphosate,bromoxynil or imidazolinone may find particular use. Such genes havebeen reported (Stalker et al., J. Biol. Chem. (1985) 260:4724-4728(glyphosate resistant EPSP); Stalker et al., J. Biol. Chem. (1985)263:6310-6314 (bromoxynil resistant nitrilase gene); and Sathasivan etal., Nucl. Acids Res. (1990) 18:2188 (AHAS imidazolinone resistancegene)).

Stable transformation of tobacco plastid genomes by particle bombardmentis reported (Svab et.al. (1990 supra) and Svab et al. (1993 supra)). Themethods described therein may be employed to obtain plants homoplasmicfor plastid expression constructs.

Generally, bombarded tissue is cultured for approximately 2 days on acell division-promoting media, after which the plant tissue istransferred to a selective media containing an inhibitory amount of theparticular selective agent, as well as the particular hormones and othersubstances necessary to obtain regeneration for that particular plantspecies. Shoots are then subcultured on the same selective media toensure production and selection of homoplasmic shoots.

Homoplasmy is verified by southern analysis. In the examples providedbelow, BamHI-digested total cellular DNA is tested with various probes,specifically, a part of the plastid targeting fragment, an aadAfragment, a 1.8 kb cry1A fragment and a 3.5 kb fragment of the cry73coding region. Southern blot analysis with these probes confirms theintegration of the chimeric cry genes in the tobacco plastid genome toyield transplastome lines.

As an alternative to a second round of shoot formation, the initialselected shoots may be grown to mature plants and segregation reliedupon to provide transformed plants homoplastic for the inserted geneconstruct.

Where transformation and regeneration methods have been adapted for agiven plant species, either by Agrobacterium-mediated transformation,bombardment or some other method, the established techniques may bemodified for use in selection and regeneration methods to produceplastid-transformed plants. For example, the methods described hereinfor tobacco are readily adaptable to other solanaceous species, such astomato, petunia and potato.

In Brassica, Agrobacterium-mediated transformation and regenerationprotocols generally involve the use of hypocotyl tissue, a non-greentissue which might contain a low plastid content. Thus, for Brassica,preferred target tissues would include microspore-derived hypocotyl orcotyledonary tissues (which are green and thus contain numerousplastids) or leaf tissue explants. While the regeneration rates fromsuch tissues may be low, positional effects, such as seen withAgrobacterium-mediated transformation, are not expected, thus it wouldnot be necessary to screen numerous successfully transformed plants inorder to obtain a desired phenotype.

The vectors for use in plastid transformation preferably include meansfor providing a stable transfer of the plastid expression construct andselectable marker construct into the plastid genome. This is mostconveniently provided by regions of homology to the target plastidgenome. The regions of homology flank the construct to be transferredand provide for transfer to the plastid genome by homologousrecombination, via a double crossover into the genome. The complete DNAsequence of the plastid genome of tobacco has been reported (Shinozakiet al. (1986) EMBO J. 5:2043-2049). Complete DNA sequences of theplastid genomes from liverwort (Ohyama et al. (1986) Nature 322:572-574)and rice (Hiratsuka et al. (1989) Mol. Gen. Genet. 217:185-194), havealso been reported.

Where the regions of homology are present in the inverted repeat regionsof the plastid genome (known as IRA and IRB), two copies of thetransgene are expected per transformed plastid. The regions of homologywithin the plastid genome are approximately 1 kb in size. Smallerregions of homology may also be used, and as little as 100 bp canprovide for homologous recombination into the plastid genome. However,the frequency of recombination and thus the frequency of obtainingplants having transformed plastids decreases with decreasing size of thehomology regions.

Examples of constructs having regions of homology the plastid genome aredescribed in Svab et.al. (1990 supra) and Svab et al. (1993 supra).Regions useful for recombination into tobacco and Brassica plastidgenomes are also identified in the following examples, but homologousrecombination and selection constructs may be prepared using manyplastid DNA sequences, and to any target plant species. In the examplesprovided herein, the flanking tobacco plastid homology regions of theplastid expression construct direct the insertion of a Bacillusthuringiensis transgene into the tobacco genome between trnv and therps12 operon. Since integration into the plastid genome occurs byhomologous recombination and the target site is in an inverted repeatregion of the plastid genome, two copies of the transgene per plastidgenome are expected. Selection is made for the spectinomycin resistancemarker phenotype expressed by the aadA gene.

In the examples the native cry gene, i.e., having an unmodified codingregion to the protoxin, is placed into a plastid expression constructfor expression of Bacillus thuringiensis toxin from the plant plastid.

A synthetic Bacillus thuringiensis gene is placed in the same expressionconstruct as the protoxin gene. The synthetic gene is designed to havetobacco RuBPCO small subunit codon usage, with an overall increase inthe guanine plus cytosine content to 55% (with respect to the nativegene content of 39%), and has been truncated to leave only thosesequences which enode the active fragment of the toxin. Such a gene isknown to provide optimal expression from the plant nuclear genome. Boththe bacterial gene which has been resynthesized for increased expressionfrom plant nuclear transformation and the non-resynthesized,non-truncated wild-type gene to the protoxin are introduced via achloroplast transformation vector (FIG. 1).

Unexpectedly, it is found that expression of the toxin is greatlyenhanced from the native encoding sequence for the gene, as opposed to aversion of the gene resynthesized to approximate the preferred codons ofthe plant genome. Tobacco lines containing the native encoding sequencedemonstrate strong insecticidal bioactivity, as measured by insectfeeding assays. Tobacco lines having a synthetic cryIA(c) genedemonstrate no observable bioactivity. As in both cases the constructsare introduced in a controlled manner by homologous recombination fromthe same plastid vector, the differences cannot be accounted for bypositional effects.

In transformed plants containing the native encoding sequence, theBacillus thuringiensis toxin is present as a component of up to about 5%or greater of the total leaf protein, a level which is much higher thanis present in the leaf of plants resulting from nuclear transformation.In plants containing the gene resynthesized to approximate the preferredcodons of the plant genome, the MRNA to the toxin appears degraded, andlittle or no toxin protein appears present in the leaf.

That a native Bacillus thuringiensis toxin gene is expressed to such ahigh level in the plastid, while an otherwise identical constructcontaining a Bacillus thuringiensis gene resynthesized for efficientnuclear expression is very poorly expressed in the plastid, despitehaving the same copy number in the plastid, suggests that the adenineplus thymine content of the plastid transgene heavily influencesexpression. As previously noted, the guanine plus cytosine content ofthe synthetic gene was increased to 55%, which is high relative to thatof the plastid genome content of less than 40% guanine plus cytosine.This difference may cause inefficient processing of the mRNA, or lead toan increase in its rate of degradation. The native Bacillusthuringiensis gene has a guanine plus cytosine percentage which moreclosely matches that of the plastid genome, and thus more closely favorsthe codon usage of a plastid gene.

The adenine plus thymine content of the respective genes may notentirely explain the dramatic differences in expression of the nativeand synthetic Bacillus thuringiensis toxin proteins. One additionalfactor which could be postulated is that unwanted or highly inefficientplastid RNA processing signals are introduced into the syntheticczyIA(c) gene. Such signals, if present, could greatly reduce or eveneliminate expression of the toxin.

In any case, it is now shown that the codon usage of the native Bacillusthuringiensis gene achieves an expression level which is much higher inplastid expression than is possible with resynthesized sequence to thesame gene, thus demonstrating that a gene having bacterial codon usagecan achieve high levels of expression in a plant plastid. The aboveresults eliminate the need to resynthesize a certain class of genes forhigh level expression in plants.

The DNA sequence of interest may have a natural codon usage high inadenine and thymine, as is the case for the Bacillus thuringiensis gene,or may alternatively be resynthesized to enrich the adenine plus thyminecontent. In fact, while the constructs and methods described herein maybe employed with a wide variety of native bacterial DNA encodingsequences, a wider range of potential gene targets for high levelplastid expression can be obtained by resynthesizing genes, for instanceplant nuclear genes, to increase the adenine and thymine content of theencoding sequence.

The invention now being generally described, it will be more readilyunderstood by reference to the following examples which are included forpurposes of illustration only and are not intended to limit the presentinvention.

EXAMPLES

In the experimental disclosure which follows, all temperatures are givenin degrees centigrade (°), weights are given in grams (g), milligram(mg) or micrograms (μg), concentrations are given as molar (M),millimolar (mM) or micromolar (μM) and all volumes are given in liters(l), milliliters (ml) or microliters (μl), unless otherwise indicated.

Example 1 Plastid Transformation Vectors

Constructs and methods for use in transforming the plastids of higherplants are described in Svab et al. (1990 supra), Svab et al. (1993supra) and Staub et al. (1993 supra). The complete DNA sequences of theplastid genome of tobacco are reported by Shinozaki et al. (1986 supra).All plastid DNA references in the following description are to thenucleotide number from tobacco.

The cryIA(c) gene is obtained from plasmid pBtkHD73 (Toagosei ChemicalCo., Japan). This gene is further processed by digestion with SmaI/NsiIand a synthetic adapter is inserted (top strand: (SEQ ID NO: 1)5'-CCCGGATCCATGGATAACAATCCGA-ACATCAATGAATGCA-3'; bottom strand; (SEQ IDNO: 2) 5'-TTCATTGATGTTCGGATT-GTTATCCATGGATCCGGG-3'). The entire 5'untranslated region from the cryIA(c) gene is then removed, and an NcoIsite is introduced at the natural start codon (position 163 of thenucleotide sequence (Adang et al. (1985) Gene 36;289-300). A BamHI siteis introduced just upstream of the NcoI site. Oligonucleotidemutagenesis is performed to introduce BglII and SalI sites directlyadjacent to the stop codon of the cryIA(c) gene, to facilitate removalof unwanted DNA 3' of the coding region. The remaining sequence includesthe entire encoding region to the protoxin.

A synthetic cryIA(c) gene encoding the active toxin fragment isconstructed by annealing and ligating 70 and 90 base oligonucleotides,in a method as described (Wosnick et al. (1987) Gene 60;115-127). Thesynthetic gene is designed to have tobacco RuBISCO small subunit codonusage, including a guanine and cytosine content of 55%, with an NcoIsite at the start codon and a SalI site at the stop codon, while stillencoding the amino acid sequence of the toxin. This synthetic gene isalso truncated, however, so that the encoding region only provides theamino acid sequence to the active fragment of the protoxin.

A plastid transformation vector is used which carries a passenger genein a Prrn(L)rbcL(S)/Trps16 expression cassette, with polylinkerrestriction sites. The Prrn(L)rbcL(S) fragments are described in Svab etal. (1993 supra). To further secure the stability of the mRNAs, theTrps16 fragment is cloned downstream of the passenger gene encodingregion. The Trps16 fragment comprises the rps16 gene 3'-regulatoryregion from nucleotides 5,087 to 4,939 in the tobacco plasmid DNA.

Chimeric genes are preferably inserted into the vector to direct theirtranscription towards the rrn operon. Thus, in the plastid genome,chimeric genes are transcribed from the Prrn(L)rbcL(S) 5'-regulatoryregion comprising the long rrn operon promoter fragment from nucleotides102,561 to 102,677 of the tobacco plastid genome, which is fused with asynthetic leader sequence designed after the rbcL gene leader betweennucleotides 57,569 to 57,584 in the plastid DNA.

The plastid transformation vector also carries a selectablespectinomycin resistance gene (aadA) under control of psbA geneexpression signals. The regulatory and encoding sequences are alsoflanked by plastid DNA homology regions whose limits are bp 138,447(EcoRI) to 140,219 (HincII) and 140,219 (HincII) to 141,382 (BglII) ofthe tobacco plastid genome (Shinozaki et al. (1986 supra)). This directsinsertion of foreign genes located between the flanking regions into theplastid between the trnV gene and the rps12/7 operon.

This plastid transformation vector is digested with the NcoI/SalIrestriction endonucleases to remove the encoding region of the passengergene, which is then replaced with a NcoI/SalI fragment containing thesynthetic cryIA(c) coding region, yielding a vector which is designatedpZS223 (FIG. 1). The wild type cryIA(c) protoxin gene is similarlycloned as an NcoI/SalI fragment, yielding a plasmid designated pZS224.By this approach Bacillus thuringiensis DNA 3' of the protein codingregion is omitted for both plasmids, pZS223 and pZS224.

The insertion of the respective cry genes from vectors pZS223 and pZS224into the wild-type plastid genome (Nt-ptDNA) to yield transplastomesNt-pZS223 and Nt-pZS224, respectively, is shown in FIG. 1. Theabbreviations used in FIG. 1 are as follows: 16S, 16S rRNA gene; trnV,trnV gene; aada, spectinomycin resistance gene; cry1A and cry73 aresynthetic and native Bacillus thuringiensis d-endotoxin genes,respectively. The restriction endonuclease cleavage sites are designatedas follows: B, BamHI; Bg, BglII; H, HindIII; N, NcoI; RI, EcoRI, RV,EcoRV; S, SalI.

Example 2 Plant Plastid Transformation

Stable transformation of tobacco plastid genomes by particle bombardmentis reported in Svab et.al. (1990 supra) and Svab et al. (1993 supra).The methods described therein may be employed to obtain plantstransformed with the plastid expression constructs described herein.Such methods generally involve DNA bombardment of a target host explant,preferably an explant made from a tissue which is rich in metabolicallyactive plastids, such as green plant tissues including leaves orcotyledons.

Tobacco seeds (N. tabacum v. Xanthi N/C) are surface sterilized in a 50%chlorox solution (2.5% sodium hypochlorite) for 20 minutes and rinsed 4times in sterile H₂ O. These are plated asceptically on a 0.2×MS saltsmedia and allowed to germinate. The seedlings are grown on agarsolidified MS media with 30 g/l sucrose (Murashige et al. (1962)Physiol. Plant 15:493-497).

Tungsten microprojectiles (1.0 μM) are coated with plasmid DNA accordingto Maliga (Maliga, P. (1993) Methods in Plant Molecular Biology--ALaboratory Manual, eds. Pal Maliga, Daniel Klessig, Anthony Cashmore,Wilhelm Gruissem and Joseph Varner; Cold Spring Harbor Press) and usedto bombard mature leaves, placed abaxial side up on RMOP media; MSsalts, 1 mg/l BAP, 0.1 mg/l NAA, 30 g/l sucrose and 0.7% phytagar. Svabet al. (1990) Proc. Natl. Acad. Sci. USA 87:8526-8530 (using the Bio-RadPDS 1000 He system (Sanford et al., An improved, helium-driven Biolisticdevice, Technique 3:3-16)). Plasmids pZS223 and pZS224 are used as thecoating plasmid DNA.

The bombarded tissue is then cultured for approximately 2 days on a celldivision-promoting media, after which the plant tissue is transferred toa selective media containing an inhibitory amount of the particularselective agent. Transformed explants form green shoots in approximately3-8 weeks. Leaves from these shoots are then subcultured on the sameselective media to ensure production and selection of homoplasmicshoots.

Example 3 DNA Gel Blot Analysis of Transplastomic Lines

Transformed plants selected for marker aadA marker gene expression areanalyzed to determine whether the entire plastid content of the planthas been transformed (homoplastic transformants). Typically, followingtwo rounds of shoot formation and spectinomycin selection, approximately50% of the transgenic plantlets which are analyzed are homoplastic, asdetermined by Southern blot analysis of plastid DNA. Homoplasmicplantlets are selected for further cultivation.

Following a second round of shoot formation and spectinomycin selection,two transplastomic lines for each construct are obtained, Nt-pZS223 andNt-pZS224. These lines are checked for homoplasmy. Southern blotanalysis is used to confirm the integration of the chimeric cry genes inthe tobacco plastid genome. Preparation, electrophoresis, and transferof DNA to filters is as described (Svab et al., (1993 supra)).

The complete disappearance of the 3.3 Kb native tobacco BamHI fragmentin the Nt-pZS223 and Nt-pZS224 transformants with a probe covering theregion of integration, and the appearance of expected sized bands forthe inserted DNA fragments in those transformants, 5.5 kb and 7.3 kb,respectively (see FIG. 1), establishes that the transformed plants arehomoplasmic for the intended constructs. Probing identical filters withthe aada, cryIA(c) protoxin, and synthetic cryIA(c) genes demonstrated alinkage of the aadA and cryIA(c) genes to the expected 5.5 and 7.3 KbBamHI fragments as well as the lack of these genes in the negativecontrol.

Example 4 Insect Bioassays

As described, the development of transformed plant lines NtpZS223 andNt-pZS224 is accomplished on RMOP media supplemented with 500 mg/lSpectinomycin dihydrochloride. Plants are subcloned on the sameselective medium, by the method according Svab et al.(1990 supra).Selected plants are then rooted in MS media containing 1 mg/l IBA, 500mg/l Spectinomycin dihydrochloride and 0.6% phytagar.

Helicoverpa zea and Heliothis virescens eggs are obtained from theUSDA-ARS in Stoneville, Miss. and allowed to hatch. Neonate larva areplaced on Tobacco Budworm Diet from Bioserve (Frenchtown, N.J.), andincubated in a 16:8 photoperiod at 28° C. for 5 days. The larva developduring this time to late second or early third instar.

At 5 days, fully expanded leaves are excised from the tobacco plants andplaced on 3 ml of 2% agar in a 32 well rearing tray from CDInternational (Pitman, N.J.). The larva are placed 1 per well, sealedand incubated for 5 days at the same conditions. At day 10, the leafmaterial consumed by the insect is estimated and insects checked formortality. The larva are considered dead if they showed no movementafter prodding with forceps.

Example 5 Insecticidal Feeding Activity

To determine the presence and relative amount of active Bacillusthuringiensis d-toxin in the tobacco lines homoplasmic for nativeprotoxin and synthetic `truncated` cryIA(c) gene expression constructs,efficacy of these plants to third instar Heliothis virescens (tobaccobudworm) and Helicoverpa zea (corn earworm/cotton bollworm) larvae istested (See table). Both test insects are sensitive to the cryIA(c)toxin with H. zea being 10-fold more resistant than H. virescens(MacIntosh et al. (1990) J. Invertebr. Pathol. 56:258-266.).

Third instar larvae are chosen for the bioassay since the insects aremore resistant to the toxin at this stage than are first instar larvaethus allowing a more stringent comparison between the control and testplants. Tobacco lines designated 4083 and 4084, derived by nucleartransformation with the same synthetic cryIA(c) gene as used in pZS223and shown to be highly toxic to third instar H. virescens larvae, areused as positive controls in the bioassay. Nicotiana tabacum var.`Petite Havana` serves as the negative control since this is the geneticbackground used to generate the transplastomic lines.

Table 1 is a summary of Bacillus thuringiensis tobacco insect feedingassays. The data demonstrates that transplastomic line Nt-pZS224 is verytoxic to both H. virescens and H. zea as it causes 100% mortality tothese insects while sustaining less than 2% total leaf damage. Thisresult compares favorably to the results for positive control 4083 and4084 tobacco plants. The 4083-2-4 plant when assayed with H. zea causes100% mortality but sustains a much greater level of leaf feeding damagethan the Nt-pZS224 tobacco line indicating less toxin production.Tobacco line 4084-4-1 performed comparably to Nt-pZS224 tobacco infeeding, although it does not compare to the levels of toxin produced inNt-pZS224 when measured as a component of total leaf protein.

Tobacco line Nt-pZS223 shows no detectable bioactivity.

                                      TABLE 1    __________________________________________________________________________    SUMMARY OF BT TOBACCO INSECT FEEDING ASSAYS                     plants               Vector                     tested                          Heliothis virescens                                   % Leaf eaten                                         Hetiocoverpa zea                                                  % Leaf eaten    __________________________________________________________________________    Chloroplast    synthetic toxin gene               pZS223                     223-3                          NO mortality                                   100%  NO mortality                                                  100%                     223-5                          NO mortality                                   75%   NO mortality                                                  100%                     223-12                          NT             NO mortality                                                  100%                     223-13                          NO mortality                                   75%   NT*    wild type protoxin gene               pZS224                     224-5                          100% mortality                                   2%    100% mortality                                                  2%                     224-9                          100% mortality                                   2%    100% mortality                                                  2%    Nuclear Controls    synthetic toxin gene               pCGN4083                     4083-1-2                          100% mortality                                   2%    NT*                     4083-2-4                          NT             100% mortality                                                  40%               pCGN4084                     4084-8-5                          100% mortality                                   2%    NT*                     4084-1-1                          NT             100% mortality                                                  2%    Untransformed Controls                     control 1                          25% mortality                                   75%   NO mortality                                                  100%                     control 2                          NO mortality                                   100%  NT*                     control 3                          50% mortality                                   75%   NT*    __________________________________________________________________________       10 third instar larva were individually tested per plant     *NT: Plant not tested

All publications and patent applications mentioned in this specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application is specifically andindividually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claim.

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 2    (2) INFORMATION FOR SEQ ID NO: 1 :    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 40 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: synthetic oligonucleotide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1 :    CCCGGATCCATGGATAACAATCCGAACATCAATGAATGCA40    (2) INFORMATION FOR SEQ ID NO: 2 :    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 36 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: synthetic oligonucleotide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2 :    TTCATTGATGTTCGGATTGTTATCCATGGATCCGGG36    __________________________________________________________________________

What is claimed is:
 1. A construct comprising the following as operablyjoined components in the 5' to 3' direction of transcription:(a) apromoter functional in a plant plastid; (b) a DNA sequence encoding apeptide of interest; and (c) a transcription termination region thatterminates transcription in a plant plastid, wherein said DNA sequenceencoding said peptide of interest is derived from the nuclear genome ofa plant cell, and wherein said DNA sequence encodes the same amino acidsequence as the native DNA sequence but has an enriched adenine andthyrmine content of greater than 50%.
 2. The construct according toclaim 1, wherein said construct further comprises:(d) a gene encoding amarker for selection of transformed plant cells; and (e) DNA regions ofhomology to the genome of said plastid, wherein said plant cellscomprise a plastid that expresses said marker and wherein said DNAregions of homology flank said regions (a), (b), (c) and (d) of saidconstruct and provide for homologous recombination into the plastidgenome.
 3. The construct according to claim 1 wherein said plant plastidis a chloroplast.
 4. The construct according to claim 1 wherein said DNAsequence approximates the adenine and thymine content of a plant plastidgenome.
 5. The construct according to claim 2, wherein said markerconfers resistance to spectinomycin and/or streptomycin.
 6. A method forproducing a peptide of interest in a solanaceous plant cell, said methodcomprising expressing said peptide in plastids of said solanaceous plantcell by transforming said plant cell with the construct according toclaim
 1. 7. The method of claims 6, wherein said construct furthercomprises:(d) a gene encoding a marker for selection of solanaceousplant cells; and (e) DNA regions of homology to the genome of saidplastid, wherein said solanaceous plant cells comprise a plastid thatexpresses said marker, and wherein said said DNA regions of homologyflank said components (a), (b), (c) and (d) of said construct andprovide for homologous recombination into the plastid genome.
 8. Themethod according to claim 6, wherein said plastids are chloroplasts. 9.The method according to claim 7, wherein said marker confers resistanceto spectinomycin and/or streptomycin.